Reporter molecules are routinely used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology and molecular biology. For example, reporter molecules are employed in assays where the levels of the reporter molecule are due to transcription from a specific promoter linked to the reporter molecule. Reporter molecule assays can be used to study biological processes including gene expression, receptor activity, transcription factors, intracellular signaling, mRNA processing, and protein folding. Analysis of reporter molecules that are typically used in such assays includes detection of radioactive isotopes, enzyme activity, fluorescence, or luminescence.
Luminescence in biological assays typically involves the activity of a luminogenic protein. Luminogenic proteins that are useful in assay systems include, but are not limited to, Renilla luciferase, Oplophorus luciferase, Vargula (Cypridina) luciferase, Gaussia luciferase, and aequorin. In a luminescent reaction, the interaction of a luminogenic protein with an appropriate molecule, referred to as a luminophore, produces light as one of the reaction products. The quantity of light produced in the reaction can be measured. This measurement may be used qualitatively to determine if a certain substance or target of interest is or is not present in a sample. This measurement also may be used quantitatively to calculate the concentration of luminogenic protein, luminophore, and/or substance or target of interest in the reaction.
Luminescent reactions can be used to detect very small quantities of a particular analyte, the substance being identified and measured in an assay. For example, luminescent reactions can be used to detect and quantify proteases, lipases, phosphatases, peroxidases, glycosidases, various metabolites such as ATP or NADH, and reporter molecules. Luminescent reactions can also be used to detect and quantify analytes through binding interactions, such as those mediated by antibodies and nucleotide probes. Another application of luminescent reactions is bioluminescence resonance energy transfer (BRET), which can determine if two molecules are capable of binding to each other or are co-localized in a cell. Typically, luminescent reactions can be used to detect an analyte present in a sample at less than about 1×10−16 moles, often less than 1×10−19 moles.
Coelenterazines are reporter molecules known to luminesce when acted on by a wide variety of bioluminescent proteins such as marine luciferases. Examples of such marine luciferases include Renilla luciferase, Aequorin, Gaussia luciferase, and Oplophorus luciferase.